Method and apparatus for analyzing protein-protein interaction on single-molecule level within the cellular environment

ABSTRACT

A method of analyzing protein-protein interactions includes binding the first proteins to the substrate where the first proteins are tagged with the first markers which bind specifically to the biomolecules immobilized on the substrate or the first proteins bind specifically to the biomolecules immobilized on the substrate; incubating the substrate bound first proteins with cell lysate containing the second proteins which are tagged with second markers; analyzing the interactions between the first proteins and the second proteins in the cell lysate, and obtaining the first analytic value representing the kinetic picture of the interactions; incubating the substrate bound first proteins with cell lysate mixture of a cell lysate consisting of the second markers-tagged second proteins and another cell lysate comprising other proteins including unlabelled second proteins and obtaining the second analytic value; comparing and analyzing the first and the second analytic values.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/KR2012/003077 filed on Apr. 20, 2012, which claims priority toKorean Patent Application No, 10-2011-0036942 filed on Apr. 20, 2011,Korean Patent Application No. 10-2011-0088062 filed on Aug. 31, 2011,and Korean Patent Application No. 10-2011-0088084 filed on Aug. 31,2011, which applications are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method of analyzing protein-proteininteractions. More specifically, the present invention relates to amethod and an apparatus for analyzing protein protein interactions atthe single molecule level in the context of their normal intracellularenvironment, as well as the degree of the effects of different proteinson a specific protein-protein interaction in the context of the actualintracellular environment.

BACKGROUND ART

A cell maintains a life phenomenon by performing a variety of biologicalfunctions, such as gene expression, cell growth, cell cycle, metabolism,signal transduction, and the like, which are mediated by various andcomplex protein-protein interactions. Accordingly, understanding thenature and function of such intracellular protein-protein interactionshas been a major foundation of our understandings of basic cellularprocesses, and has also played an essential part in our ability todevelop new drugs and treat diseases.

A representative method of investigating protein-protein interactions invitro is an affinity chromatography method.

In the case of protein affinity chromatography, protein has to gothrough demanding and complex purifying processes. Disadvantageously,since the interactions between proteins are only assessed in vitro, thismethodology may result in false-positive results. For example, proteinscan be bound by an electrostatic interaction while they pass through acolumn.

In order to perform a quantitative measurement, a method ofinvestigating the protein-protein interactions according to conventionalart technologies analyzes the interactions in the isolation. In otherwords, the interaction of proteins of interest is assessed in theabsence of other intracellular materials by isolating and purifying eachof the proteins away from the cellular milieu and analyzing theprotein-protein interactions. Disadvantageously, these conventional arttechnologies limit, or prevent, the ability to analyze theprotein-protein interactions at the single molecular level in thecontext of the normal intracellular environment (e.g., in the presenceof other proteins, and the like).

Moreover, methods of investigating protein-protein interactionsaccording to the conventional technologies are also disadvantageousbecause they do not allow assessment of the degrees of effects thatother proteins may have on specific protein-protein interactions in thecontext of the actual intracellular environment.

SUMMARY OF THE DISCLOSURE

An object of the present invention is to provide a method and anapparatus for analyzing protein-protein interactions, which can analyzethe protein-protein interactions at a single molecule level in thecontext of the normal intracellular environment. Additionally, an objectof the invention is to also assess the degree of effects of differentproteins on specific protein-protein interactions when the differentproteins are present in the normal intracellular environment.

Accordingly, an exemplary embodiment of the present invention provides amethod of analyzing interactions between first proteins and secondproteins at a single molecule level, comprising: (a) binding the firstproteins to the substrate where the first proteins are tagged with thefirst markers which bind specifically to the biomolecules immobilized onthe substrate; (b) incubating the substrate hound first proteins withcell lysate containing the second proteins which are tagged with secondmarkers; and (c) analyzing the interactions between the first proteinsand the second proteins in the cell lysate.

According to another aspect of the present invention, there is provideda method of analyzing interactions between first proteins and secondproteins at a single molecular level, comprising: (a) binding the firstproteins to the substrate where first proteins bind specifically to thebiomolecules immobilized on the substrate; (b) incubating the substratebound first proteins with cell lysate containing the second proteinswhich are tagged with second markers; and (c) analyzing the interactionsbetween the first proteins and the second proteins in the cell lysate.

According to a preferred embodiment, step (c) includes measuring afluorescent signal having a specific wavelength generated by the markersincluded in the second proteins bound to the first proteins using anoptical apparatus generating a near-field. Preferably, in the step (c),the fluorescent signal having the specific wavelength is cumulativelymeasured for a predetermined time period. Preferably, in the step (c),the fluorescent signal having the specific wavelength is measured inreal time under the presence of the cell lysate supplied to thesubstrate.

Preferably, the step (a) includes supplying the cell lysate comprisingthe first proteins.

Preferably, the method further comprises supplying a buffer solution tothe substrate before the step (b).

Preferably, the first markers and the second markers are fluorescentproteins having different wavelengths.

According to yet another aspect of the present invention, there isprovided a method of analyzing interactions between first proteins andsecond proteins at a single molecular level, comprising: (a) binding thefirst proteins to the substrate where the first proteins are tagged withthe first markers that bind specifically to the biomolecules immobilizedon the substrate; (b) incubating the substrate hound first proteins withcell lysate containing the second proteins which are tagged with thesecond markers; (c) analyzing the interactions between the firstproteins and the second proteins in the cell lysate, and obtaining thefirst analytic value representing the kinetic picture of theinteractions; (d) incubating the substrate bound first proteins fromstep (a) with a cell lysate mixture of a cell lysate from the step (b)and another cell lysate having other proteins; (e) and analyzing theinteractions between the first proteins and the second proteins in thepresence of other proteins from the cell lysate mixture, and obtainingthe second analytic value representing the kinetic picture of theinteractions; and (f) comparing and analyzing the first and the secondanalytic values.

According to still another aspect of the present invention, there isprovided a method of analyzing interactions between first proteins andsecond proteins at a single molecular level, comprising: (a) binding thefirst proteins to the substrate where the first proteins bindspecifically to the biomolecules which are immobilized on the substrate;(b) incubating the substrate bound first proteins with cell lysatecontaining the second proteins which are tagged with the second markers;(c) analyzing the interactions between the first proteins and the secondproteins in the cell lysate, and obtaining the first analytic valuerepresenting the kinetic picture of the interactions; (d) incubating thesubstrate bound first proteins from step (a) with a cell lysate mixtureof a cell lysate from the step (b) and another cell lysate having otherproteins; (e) and analyzing the interactions between the first proteinsand the second proteins in the presence of other proteins from the celllysate mixture, and obtaining the second analytic value representing thekinetic picture of the interactions; and (f) comparing and analyzing thefirst and the second analytic values.

Preferably, the step (c) includes measuring a fluorescent signal havinga specific wavelength generated by the second markers tagged on secondproteins when they are bound to the first proteins. Here, an opticalapparatus generating a near-field is used.

Preferably, in the step (c), the fluorescent signal having the specificwavelength is cumulatively measured for a predetermined time period.

Preferably, in the step (c), the fluorescent signal having the specificwavelength is measured in real time under the presence of the celllysate supplied to the substrate.

Preferably, the step (a) includes supplying the cell lysate comprisingthe first proteins.

Preferably, the method further comprises supplying a buffer solution tothe substrate before the step (b).

Preferably, the first markers and the second markers are fluorescentproteins having different wavelengths.

According to yet still another aspect of the present invention, there isprovided an apparatus of analyzing interactions between first proteinsand second proteins at a single molecular level, comprising: a sampleloading unit for loading a substrate comprising attached biomoleculeswhich specifically bind to first markers tagged on the first proteins,wherein the first proteins are supplied to the substrate to inducebinding of the biomolecules and the first markers, and cell lysatecontaining the second markers-tagged second proteins are supplied to thesubstrate; an optical excitation unit for generating a near-field havinga first wavelength to a surface of the substrate loaded on the sampleloading unit; and an optical measuring unit for detecting a change fromthe first wavelength to a second wavelength on the surface of thesubstrate according to the interactions between the first proteins andthe second proteins in the cell lysate.

According to yet still another aspect of the present invention, there isprovided an apparatus of analyzing interactions between first proteinsand second proteins at a single molecular level, comprising: a sampleloading unit for loading a substrate comprising attached biomoleculeswhich specifically bind to the first proteins, wherein the firstproteins are supplied to the substrate and binds to the biomolecules,and cell lysate containing the second markers-tagged second proteins aresupplied to the substrate; an optical excitation unit for generating anear-field having a first wavelength to a surface of the substrateloaded on the sample loading unit; and an optical measuring unit fordetecting a change from the first wavelength to a second wavelength onthe surface of the substrate according to the interactions between thefirst proteins and the second proteins in the cell lysate.

According to a preferred embodiment, the optical measuring unitcumulatively measures a fluorescent signal having the second wavelengthfor a predetermined time period.

Preferably, the optical measuring unit measures a fluorescent signalhaving the second wavelength in real time under the presence of the celllysate.

Preferably, the first markers and the second markers are fluorescentproteins having different wavelengths.

Preferably, the apparatus further comprises a signal analyzing unit foranalyzing the fluorescent signal having the second wavelength measuredby the optical measuring unit through image processing.

According to yet still another aspect of the present invention, there isprovided air apparatus of analyzing interactions between first proteinsand second proteins at a single molecular level, comprising: a sampleloading unit for loading a substrate comprising attached biomoleculeswhich specifically hind to first markers tagged on the first proteins,wherein the first proteins are supplied to the substrate to inducebinding of the biomolecules and the first markers, and cell lysatecontaining the second markers-tagged second proteins are supplied to thesubstrate; an optical excitation unit for generating a near-field havinga first wavelength to a surface of the substrate loaded on the sampleloading unit; an optical measuring unit for detecting a change from thefirst wavelength to a second wavelength on the surface of the substrateaccording to the interactions between the first proteins and the secondproteins in the cell lysate; and a signal analyzing unit for obtainingthe first analytic value representing the kinetic picture of theinteractions by analyzing a fluorescent signal having the secondwavelength measured by the optical measuring unit through imageprocessing; wherein a new substrate comprising attached biomoleculeswhich specifically bind to first markers tagged on the first proteins isloaded on the sample loading unit, and cell lysate mixture of a celllysate consisting of the second markers-tagged second proteins andanother cell lysate having other proteins are supplied to the newsubstrate, such that the optical measuring unit detects a change fromthe first wavelength to the second wavelength on the surface of thesubstrate according to interactions between the first proteins and thesecond proteins in the supply of the cell lysate mixture to the newsubstrate, and the signal analyzing unit obtains a second analytic valueby analyzing a fluorescent signal having the second wavelength measuredby the optical measuring unit through image processing.

According to yet still another aspect of the present invention, there isprovided an apparatus of analyzing interactions between first proteinsand second proteins at a single molecular level, comprising: a sampleloading unit for loading a substrate comprising attached biomoleculeswhich specifically bind to the first proteins, wherein the firstproteins are supplied to the substrate and binds to the biomolecules,and cell lysate containing the second markers-tagged second proteins aresupplied to the substrate; an optical excitation unit for generating anear-field having a first wavelength to a surface of the substrateloaded on the sample loading unit; an optical measuring unit fordetecting a change from the first wavelength to a second wavelength onthe surface of the substrate according to the interactions between thefirst proteins and the second proteins in the cell lysate; and a signalanalyzing unit obtains a second analytic value by analyzing afluorescent signal having the second wavelength measured by the opticalmeasuring unit through image processing; wherein a new substratecomprising attached biomolecules which specifically bind to firstmarkers tagged on the first proteins is loaded on the sample loadingunit, and cell lysate mixture of a cell lysate consisting of the secondmarkers-tagged second proteins and another cell lysate having otherproteins are supplied to the new substrate, such that the opticalmeasuring unit detects a change from the first wavelength to the secondwavelength on the surface of the substrate according to interactionsbetween the first proteins and the second proteins in the supply of thecell lysate mixture to the new substrate, and the signal analyzing unitobtains a second analytic value by analyzing a fluorescent signal havingthe second wavelength measured by the optical measuring unit throughimage processing.

According to a preferred embodiment, the apparatus further comprising asignal diagnosing unit for comparing and analyzing the first analyticvalue and the second analytic value.

Preferably, the optical measuring unit cumulatively measures thefluorescent signal having the second wavelength for a predetermined timeperiod.

Preferably, the optical measuring unit measures in real time under thepresence of the cell lysate supplied to the substrate.

Preferably, the first marker and the second marker are fluorescentproteins having different wavelengths.

According to the present invention, it is possible to analyzeprotein-protein interactions at the single molecule level in the contextof their normal intracellular environment, as well as the degree of theeffects of different proteins on a specific protein-protein interactionin the context of the actual intracellular environment.

BRIEF DESCRIPTION OF DRAWINGS

The above and other objects, features and advantages of the presentinvention will become more apparent to those of ordinary skill in theart by describing in detail exemplary embodiments thereof with referenceto the attached drawings, in which:

FIG. 1 is a flowchart illustrating a procedure of a method of analyzingprotein-protein interactions according to an exemplary embodiment of thepresent invention;

FIG. 2 to FIG. 4 are diagrams illustrating each step of a method ofanalyzing the protein-protein interactions according to an exemplaryembodiment of the present invention;

FIG. 5 is a flowchart illustrating a procedure of a method of analyzingthe protein-protein interactions according to another exemplaryembodiment of the present invention;

FIG. 6 is a function block diagram showing an exemplary structure of anapparatus performing a method of analyzing the protein-proteininteractions in FIG. 1 to FIG. 5; and

FIG. 7 is a diagram showing a signal measured by an optical measuringunit of the apparatus of analyzing the protein-protein interactionsaccording to an exemplary embodiment of the present invention.

DETAILED DESCRIPTION

Hereinafter, exemplary embodiments of the present invention will bedescribed in detail. However, the present invention is not limited tothe embodiments disclosed below, but can be implemented in variousforms. The following embodiments are described in order to enable thoseof ordinary skill in the art to embody and practice the presentinvention.

With reference to the appended drawings, exemplary embodiments of thepresent invention will be described in detail below. To aid inunderstanding the present invention, like numbers refer to like elementsthroughout the description of the figures, and the description of thesame elements will be not reiterated.

FIG. 1 is a flowchart illustrating a method of analyzing protein-proteininteractions according to an exemplary embodiment of the presentinvention. Referring to FIG. 1, an analyst performs an expression offirst fluorescent protein tags, a marker of first proteins, on the firstproteins (i.e. a first protein having a first marker) in relevant cellsthrough genetic manipulation, in order to analyze the interactionbetween first proteins and second proteins at the single molecule level(S110). According to an exemplary embodiment of the present invention,the first fluorescent proteins may be attached or connected to the firstproteins by a physical-chemical method.

According to an exemplary embodiment of the present invention, the firstproteins are h-Ras proteins, the second proteins are Ras-binding domain(RBD) proteins of C-Raf, and the first fluorescent proteins are m-Cherryproteins.

Subsequently; the analyst attaches first fluorescent protein-bindingantibodies that are to be bound to the first fluorescent proteins taggedas the markers to the first proteins in the above-described step, S110,to a substrate that is a quartz slide coated with polyethylene glycol(PEG), as described in FIG. 2 (S120).

According to an exemplary embodiment of the present invention,biomolecules binding to the first fluorescent proteins, such as DNA,RNA, liposome having specific components to be hound to proteins, andthe like, may also be used in addition to the antibodies asalternatives.

The analyst then induces (S140) a binding of the first fluorescentprotein-binding antibodies and the first fluorescent protein tags, thefirst markers, on first proteins by supplying (S130) a cytoplasmiclysate of the cell, in which the first fluorescent proteins are taggedto the first proteins, prepared in the above-described step, S110, tothe substrate (S140).

Meanwhile, according to the above-described step, S130, cell lysate, forexample, cytolysate, diluted cytoplasmic lysate, diluted cytolysate, andthe like, may be used in addition to the cytoplasmic lysate.

As shown in FIG. 3, the analyst can confirm whether the firstfluorescent proteins are bound to a plurality of the first fluorescentprotein-binding antibodies attached on the substrate from a change thewavelength emitted by the first fluorescent proteins (i.e., measuringindividual single molecular signals generated from the first fluorescentproteins) by performing an observation of a substrate surface using atotal internal reflection fluorescence microscope when the firstfluorescent proteins are bound to the first fluorescent protein-bindingantibodies attached on the substrate.

According to an exemplary embodiment of the present invention, theprotein tags, the first markers, on first proteins which bind to theantibodies are not always certain to be fluorescent proteins, but theprotein tags on first proteins may be preferably the fluorescentproteins because, when they are not the fluorescent proteins, it cannotbe confirmed whether or not they are bound to antibodies through thetotal internal reflection fluorescence microscope.

In other words, the predetermined protein tags on the first proteins,the first markers, according to the present invention, function asantigens to be bound to the antibodies attached on the substrate.Therefore, the first markers, the predetermined antigens, attached tothe first proteins as described above are not always required even to beproteins, but they may also be compounds which are bound to antibodies.

That is, since when the protein tags on the first proteins are thefluorescent proteins, whether they are bound to antibodies or not can beconfirmed with the total internal reflection fluorescence microscope,and thus the number of the fluorescent proteins bound to the antibodiesattached on the substrate and binding density thereof can be accuratelymeasured.

Once the first fluorescent proteins are hound to a plurality of firstfluorescent protein-binding antibodies attached to the substrate,respectively, the analyst removes the remaining materials included inthe cytoplasmic lysate except the first proteins tagged with firstfluorescent proteins from the substrate by supplying a buffer solutionto the substrate (S150).

Subsequently, the analyst performs an expression of second fluorescentprotein tags, a marker of the second proteins, on the second proteins(i.e. second proteins having second marker) in relevant cells that arethe same type as the cells used in the above-described step, S110 (S160)through genetic manipulation of the second proteins. According to anembodiment of the present invention, the second fluorescent proteins maybe attached or connected to the second proteins by a physical-chemicalmethod. In a preferred embodiment of the invention, the second markeremits fluorescence at a different wavelength than the first marker.

The above-described step, S160, may be performed in advance along withthe above-described step, S110, in order to smoothly continue theanalysis. According to an exemplary embodiment of the present invention,since the second fluorescent proteins preferably have a wavelength rangedifferent from the first fluorescent proteins, when the firstfluorescent proteins are m-Cherry proteins, the second fluorescentproteins may be chosen as enhanced Green Fluorescent Protein (eGFP) thatis a green fluorescent protein.

The analyst supplies the whole cytoplasmic lysate of the cell with theexpressed second fluorescent proteins tags on the second proteins in theabove-described step, S160, to the substrate (S170).

The above-described step, S170, cell lysate, such as cytolysate, dilutedcytoplasmic lysate, diluted cytolysate, and the like, may be used inaddition to the cytoplasmic lysate.

The first proteins are bound to a plurality of first fluorescentprotein-binding antibodies attached to the surface of the substratethrough the first fluorescent proteins, respectively. When the wholecytoplasmic lysate including the second proteins is supplied to thesurface of the substrate as mentioned above, the first proteins on thesurface of the substrate interact with the second proteins under thesame conditions as the intracellular environment, which allows for theco-existence of the second proteins included in the cytoplasmic lysatewith native proteins in whole cell lysate.

More specifically, as shown in FIG. 4, each of the first proteins boundto each of the antibodies attached on the surface of the substratethrough the first fluorescent proteins may bind and unbind with thesecond protein repeatedly at the single molecule level under the sameconditions as the intracellular environment to repeat the binding andunbinding.

As shown in FIG. 4, when binding occurs between the first proteins andthe second proteins at the single molecule level, the analyst canconfirm that the wavelength at the surface of the substrate is changedfrom 473 nm to 520 nm by eGFP, e.g. the second fluorescent protein tagson the second proteins by observing the surface of the substrate throughan optical apparatus that can generate a near-field, such as the totalinternal reflection fluorescence microscope having a wavelength of 473nm.

In other words, the binding/unbinding state between the first proteinsand the second proteins can be confirmed by a detection of fluorescencesignals of a specific wavelength bandwidth (520 nm) generated from thesecond fluorescent proteins (eGFP) near the surface of the substratethrough the binding between the first proteins and the second proteins,and the analyst can analyze the interactions, such as the frequencies ofthe binding and unbinding between the first proteins and the secondproteins, and the like, at the single molecule level by continuouslyobserving the change of wavelength at each of binding/unbindingpositions on the substrate (S180).

In addition, the interaction, such as the frequencies of the binding andunbinding between the first proteins and the second proteins, and thelike, can be analyzed in an environment equivalent to the normalintracellular by measuring the fluorescence signals having a specificwavelength in real time under the presence of the cytoplasmic lysatesupplied to the substrate in the above-described step, S170.

It is possible that the second proteins that are not bound to the firstproteins may be moved closer to the surface of the substrate in theprocess of floating and moving in the cytoplasmic lysate supplied to thesubstrate. Consequently, this may introduce errors into the analysis ofthe protein-protein interactions caused by changing the wavelength from473 nm to 520 nm on the surface of the substrate by eGFP, i.e., thesecond fluorescent protein tags on second proteins when observing thesurface through the total internal reflection fluorescence microscope.

Therefore, according to an embodiment of the present invention, when thechange of wavelength is measured on the surface of the substrate throughthe total internal reflection fluorescence microscope, the change ofwavelength may be preferably cumulatively measured for a predeterminedtime period.

Since the rate of the movement of the second proteins that are not boundto the first proteins floating in the cytoplasmic lysate are very fast,although the second proteins temporarily move near the surface of thesubstrate during floating and moving, they leave the surface of thesubstrate again during the time of cumulatively measuring.

To verify this, it was experimentally confirmed that the wavelength isnot changed when change of wavelength is cumulatively measured on thesurface of the substrate in an integral section of, for example, 50msec. Here, all of the second proteins in the cytoplasmic lysate werefloating and moving without binding to the first proteins by, forexample, mutating proteins not to bind each others.

According to an exemplary embodiment of the present invention, the valueof analyzing protein-protein interactions in the above-described step,S180, is secured as a first analytic value and the value of analyzingprotein-protein interactions in the following different environment issecured as a second analytic value, which allows the comparativeanalysis of both values.

That is, the analyst repeats equally S110 to S160 as described aboveafter securing the value of analyzing the interaction in theabove-described step, S180, as the first analytic value.

In order to perform the above-described step, S170, the analyst suppliesa mixture of s cytoplasmic lysate and the cytoplasmic lysate from, forexample, a tumor cell, the like, and the cytoplasmic lysate of therelevant cell which does not contain the second fluorescentproteins-tagged second proteins in the above-described step, S160, tothe substrate (S190).

In order to perform the above-described step, S190, mixed cell lysate,such as mixed cytolysate, diluted mixed cytoplasmic lysate, dilutedmixed cytolysate, and the like, may be used in addition to the mixedcytoplasmic lysate.

The first proteins are bound to a plurality of first fluorescentprotein-binding antibodies attached on the surface of the substratethrough the first fluorescent proteins, respectively. When the mixtureof a cytoplasmic lysate of interest and the cytoplasmic lysate includingthe second proteins is supplied to the surface of the substrate, thisallows the first proteins on the surface of the substrate to interactnot only with second fluorescence protein tagged second proteins in thecytoplasmic lysate but also with coexisting unlabeled second proteinsand native proteins in the whole cell lysate.

In other words, unlike the above-described step, S180, the secondproteins tagged with second fluorescent proteins interact with the firstproteins on the surface of the substrate in competition with coexistingsecond proteins without second fluorescent proteins tag in the celllysate of interest. Moreover, the interactions of the second fluorescentproteins tagged second proteins with the first proteins are affected byother proteins in the cell of interest.

The degrees of effects caused by the second proteins in the cells ofinterest or other proteins in the cells of interest as mentioned abovemay be confirmed by analyzing the interactions, such as the frequenciesof the binding and unbinding of the second proteins-tagged with secondfluorescent proteins and the first proteins on the surface of thesubstrate through an observation of the surface of the substrate usingan optical apparatus generating a near-field, such as the total internalreflection fluorescence microscope, and the like, like theabove-described step, S180, by the analyst (S195).

More specifically, the analyst can analyze the degrees of effects onpromoting or inhibiting the interactions with the second proteins inother cells and other proteins in other cells for the interactionsbetween the second proteins and the first proteins by comparing andanalyzing the second analytic value, that is the value for analyzing inthe above-described step, S195, and the first analytic value asmentioned above.

FIG. 5 is a flowchart illustrating a procedure of a method of analyzingthe protein-protein interactions according to another exemplaryembodiment of the present invention. As shown in FIG. 5, theinteractions between the first proteins and the second proteins may beanalyzed in the direct binding of the first proteins to the antibodiesattached to the substrate by attaching the first protein-bindingantibodies, not the first fluorescent protein-binding antibodies to thesubstrate and not expressing the first fluorescent proteins in the firstproteins.

Referring to FIG. 5, for the method of analyzing protein-proteininteractions according another exemplary embodiment of the presentinvention, the analyst attaches first fluorescent protein-bindingantibodies to be bound to the first proteins to a substrate that is aquartz slide coated with polyethylene glycol (PEG) in order to analyzethe interactions between the first proteins and the second proteins thatare specific two proteins at the single molecule level (S210).

According to an exemplary embodiment of the present invention,biomolecules binding to the first fluorescent proteins, such as DNA,RNA, liposome having specific components to be bound to proteins, andthe like, may also be used in addition to the antibodies asalternatives. Subsequently, the analyst induces the bindings between thefirst proteins and the first protein-binding antibodies by supplying thecytoplasmic lysate (S220) of the cell including the first proteins tothe substrate (S230).

In order to perform the above-described step, S220, cell lysate, such ascytolysate, diluted cytoplasmic lysate, diluted cytolysate, and thelike, may be used in addition to the cytoplasmic lysate.

According to another exemplary embodiment of the present invention, theanalyst may pre-treat to express the predetermined fluorescent proteintags, such as m-Cherry, on the first proteins. In this case, when thefirst protein-binding antibodies are hound to the first proteins, theanalyst may confirm whether the first proteins are bound to a pluralityof the first protein-binding antibodies attached on the substrate fromthe change of wavelength by the first fluorescent proteins tags on thefirst proteins through performing an observation of the surface of thesubstrate using the total internal reflection fluorescence microscope.

Respectively, the analyst removes the remaining materials included inthe cytoplasmic lysate except the first proteins from the substrate bysupplying a buffer solution to the substrate (S240). Subsequently, theanalyst manipulates the cells to express fluorescent protein taggedsecond proteins through genetic manipulations of second proteins indifferent cells that are the same type as the above-described cells(S250).

The above described step, S250, may be performed in advance before theabove-described step, S210, in order to smoothly continue the analysis.According to an preferred embodiment of the present invention, thefluorescent proteins that are expressed on the second proteins may beeGFP.

Subsequently, the analyst supplies the whole cytoplasmic lysate of thecell having the expressed fluorescent protein tags on second proteins tothe substrate (S260).

In order to perform the above-described step, S260, cell lysate, such ascytolysate, diluted cytoplasmic lysate, diluted cytolysate, and thelike, may be used in addition to the cytoplasmic lysate.

A plurality of the first protein-binding antibodies attached on thesurface of the substrate is hound to the first proteins, respectively.When the whole cytoplasmic lysate including the second proteins issupplied to the surface of the substrate as described above, the firstproteins on the surface of the substrate interact with the secondproteins included in the cytoplasmic lysate in the same conditions asthe intracellular environment.

In other words, each first protein bound to each antibody attached onthe surface of the substrate interacts with the second protein in thesame conditions as the normal intracellular environment so that as theydo in the cell, they are repeatedly bound and unbound at the singlemolecular level.

When the first proteins are bound to the second proteins at the singlemolecule level, the wavelength on the surface of the substrate ischanged from 473 nm to 520 nm by fluorescent protein tags, i.e., eGFPs,on the second proteins; and the analyst may confirm the bindings statebetween the first proteins and the second proteins through theabove-mentioned change of wavelength by observing the surface of thesubstrate using the total internal reflection fluorescence microscopeand may analyze the interactions, such as the frequencies of the bindingand unbinding between the first proteins and the second proteins, andthe like, by continuously observing the change of wavelength of eachantibody attached to the surface of the substrate (S270).

Moreover, for performing the method of analyzing protein-proteininteractions according to another embodiment of the present invention asshown in FIG. 5, the value of analyzing the protein-protein interactionsin the above-described step, S270, is secured as a first analytic valueand the value of analyzing the protein-protein interactions in thefollowing different environment is secured as a second analytic value,and then both of the values may be compared and analyzed.

In other words, the analyst performs the above-described steps, S210 toS250, equally and repeatedly, after securing the value of analyzing theinteractions in the above-described step, S270 as the first analyticvalue.

In order to perform the above-described step, the analyst supplies amixture of s cytoplasmic lysate and the cytoplasmic lysate from, forexample, a tumor cell, the like, and the cytoplasmic lysate of therelevant cell which does not contain the second fluorescentproteins-tagged second proteins in the above-described step, S260(S280).

For performing the above-described step, S280, mixed cell lysate, suchas mixed cytolysate, diluted mixed cytoplasmic lysate, diluted mixedcytolysate, and the like, may be used in addition to the mixedcytoplasmic lysate.

The first proteins are bound to a plurality of first fluorescentprotein-binding antibodies attached on the surface of the substratethrough the first fluorescent proteins, respectively. When the mixtureof a cytoplasmic lysate of interest and the cytoplasmic lysate includingthe second proteins is supplied to the surface of the substrate, thisallows the first proteins on the surface of the substrate to interactnot only with second fluorescence protein tagged second proteins in thecytoplasmic lysate but also with coexisting unlabeled second proteinsand native proteins in the whole cell lysate.

In other words, unlike the above-described step, S270, the secondproteins tagged with second fluorescent proteins interact with the firstproteins on the surface of the substrate in competition with coexistingsecond proteins without second fluorescent proteins tag in the celllysate of interest. Moreover, the interactions of the second fluorescentproteins tagged second proteins with the first proteins are affected byother proteins in the cell of interest.

The degrees of effects caused by the second proteins in the cells ofinterest or other proteins in the cells of interest as mentioned abovemay be confirmed by analyzing the interactions, such as the frequenciesof the binding and unbinding of the second proteins-tagged with secondfluorescent proteins and the first proteins on the surface of thesubstrate through an observation of the surface of the substrate usingan optical apparatus generating a near-field, such as the total internalreflection fluorescence microscope, and the like, like theabove-described step, S180, by the analyst (S290).

More specifically, the analyst may analyze the degrees of effects, forexample, enhancement or inhibition effects of other proteins and secondproteins inside the other cells to interactions between the secondproteins and the first proteins by comparing and analyzing the secondanalytic value that is the analytic value in the above-described step,S290, and the first analytic value as mentioned above.

Meanwhile, a method of analyzing the protein-protein interactionsaccording to the present invention as shown in FIG. 1 to FIG. 5 may beperformed in an analysis apparatus having the structure as shown in FIG.6.

Referring to FIG. 6, the analysis apparatus 300 for analyzing theprotein-protein interactions according to an exemplary embodiment of thepresent invention includes an optical excitation unit 320, a sampleloading unit 310, an optical measuring unit 330, a signal analyzing unit340, a signal diagnosing unit 360, and a storage unit 350.

Firstly, the substrate used for the method of analyzing theprotein-protein interactions in the above-described present invention isloaded on the sample loading unit 310. The optical excitation unit 320generates a near-field having a first wavelength on the surface of thesubstrate loaded on the sample loading unit 310.

The optical measuring unit 330 detects the change of wavelength from thefirst wavelength to the second wavelength on the surface of thesubstrate according to the interactions between the second proteinshaving the fluorescent proteins and the first proteins attached to thesurface of the substrate in the supply of the mixed cytoplasmic lysateor the cytoplasmic lysate to the substrate.

According to an embodiment of the present invention, when thefluorescent proteins having the second proteins are eGFP, the firstwavelength would be 473 nm and the second wavelength would be 520 nm.

Moreover, the optical measuring unit 330 may: cumulatively measure thefluorescent signal of the second wavelength for a predetermined timeperiod; also measure the fluorescent signal of the second wavelength inreal time under the presence of the cytoplasmic lysate or mixedcytoplasmic lysate on the substrate; and may preferably have singlemolecular sensitivity for measuring the same.

Meanwhile, the signal analyzing unit 340 analyzes the fluorescent signalof the second wavelength measured by the optical measuring unit 330through image processing to calculate an analytic value and then storesthe calculated analytic value in the storage unit 350.

In addition, the signal analyzing unit 340 analyzes values individuallycalculated based on the fluorescent signal of the second wavelength asmeasured by the optical measuring unit 330 in each situation, in whichvarious cytoplasmic lysate, such as the cytoplasmic lysate, mixedcytoplasmic lysate, and the like, are supplied to the substrates. Thenthe signal analyzing unit 340 stores the values in the storage unit 350.

Specifically, the signal analyzing unit 340 finds a baseline of signalmarked with a red line among the fluorescent signals measured by theoptical measuring unit 330 as shown in FIG. 7.

The baseline of signal is increased between 10 seconds and 20 seconds,which means that the cytoplasmic lysate or mixed cytoplasmic lysateincluding the second proteins arrives at an imaging region of theoptical measuring unit 330.

Meanwhile, the signal analyzing unit 340 measures a fluctuation (e.g.,Noise) generated around the increased signal baseline and then sets athreshold that defines the meaningful protein-protein interactions basedon the noise.

The noise generated around the signal baseline may be formed by themovements of the second proteins that are continuously, and quicklymoved around without interacting with the proteins and the signalanalyzing unit 340 sets the baseline as a value that cannot be exceededby the noise regardless of the above interactions.

When there is a signal that exceeds the baseline, the signal analyzingunit 340 decides that there are meaningful interactions between thefirst proteins and the second proteins at the relevant point, measuresthe time (τ_(off)) of the duration of the interactions and the time(τ_(on)) required for forming the following bindings between the firstproteins and the second proteins, and then stores the above values asthe analytic values in the storage unit 350.

The time (τ_(off)) of the duration of the interaction and the time(τ_(on)) required for forming the following interaction between thefirst proteins and the second proteins obtained from the signalanalyzing unit 340 may be analyzed to thereby obtain a series of dataregarding the protein-protein interactions. The signal diagnosing unit360 compares and analyzes the analytic values calculated in eachcondition, in which various cytoplasmic lysate are supplied to thesubstrate, and then performs the functions to deduct a biological andmedical diagnosis for each cytoplasmic condition according to apredetermined diagnosis algorithm based on the results of comparing andanalyzing.

According to the present invention, protein-protein interactions in theactual intracellular environment can be analyzed at a single moleculelevel.

In addition, according to the present invention, the degrees of effectsof other proteins on specific protein-protein interactions can beanalyzed when the other proteins are involved in the lysatecorresponding to the normal intracellular environment.

While the invention has been shown and described with reference tocertain exemplary embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the spirit and scope of the invention asdefined by the appended claims.

1. A method of analyzing protein interactions at a single moleculelevel, comprising: (a) binding the first proteins to the substrate wherethe first proteins are tagged with the first markers that bindspecifically to the biomolecules immobilized on the substrate; (b)incubating the substrate hound first proteins with cell lysatecontaining the second proteins which are tagged with the second markers;(c) analyzing the interactions between the first proteins and the secondproteins in cell lysate.
 2. A method of analyzing protein interactionsat a single molecule level, comprising: (a) binding the first proteinsto the substrate where the first proteins bind specifically to thebiomolecules immobilized on the substrate; (b) incubating the substratebound first proteins with cell lysate containing the second proteinswhich are tagged with the second markers; (c) analyzing the interactionsbetween the first proteins and the second protein in cell lysate.
 3. Themethod of claim 1 or 2, wherein step (c) includes measuring afluorescent signal having a specific wavelength generated by the secondmarkers tagged on second proteins when they are bound to the firstproteins. Here, an optical apparatus generating a near-field is used. 4.The method of claim 3 in the step (c), wherein the fluorescent signalhaving the specific wavelength is cumulatively measured for apredetermined time period.
 5. The method of claim 3 in the step (c),wherein the fluorescent signal having the specific wavelength ismeasured in real time in the cell lysate supplied to the substrate. 6.The method of claim 1 or 2, wherein the step (a) includes incubating thecell lysate comprising the first proteins.
 7. The method of claim 6,further comprising incubating the substrate with a buffer solutionbefore the step (b).
 8. The method of claim 1, wherein the first markerand the second marker are fluorescent proteins having differentwavelengths.
 9. A method of analyzing interactions between firstproteins and second proteins at a single molecule level, comprising: (a)binding the first proteins to the substrate where the first proteins aretagged with the first markers that bind specifically to the biomoleculesimmobilized on the substrate; (b) incubating the substrate bound firstproteins with cell lysate containing the second proteins which aretagged with the second markers; (c) analyzing the interactions betweenthe first proteins and the second proteins in the cell lysate, andobtaining the first analytic value representing the kinetic picture ofthe interactions; (d) incubating the substrate bound first proteins fromstep (a) with a cell lysate mixture containing a cell lysate from thestep (b) and another cell lysate having other proteins; (e) analyzingthe interactions between the first proteins and the second proteins inthe presence of other proteins from the cell lysate mixture, andobtaining the second analytic value representing the kinetic picture ofthe interactions; and (f) comparing and analyzing the first and thesecond analytic values.
 10. A method of analyzing interactions betweenfirst proteins and second proteins at a single molecule level,comprising: (a) binding the first proteins to the substrate where thefirst proteins hind specifically to the biomolecules immobilized on thesubstrate; (b) incubating the substrate hound first proteins with celllysate containing the second proteins which are tagged with the secondmarkers; (c) analyzing the interactions between the first proteins andthe second proteins in the cell lysate, and obtaining the first analyticvalue representing the kinetic picture of the interactions; (d)incubating the substrate bound first proteins from step (a) with a celllysate mixture containing a cell lysate from the step (b) and anothercell lysate having other proteins; (e) analyzing the interactionsbetween the first proteins and the second proteins in the presence ofother proteins from the cell lysate mixture, and obtaining the secondanalytic value representing the kinetic picture of the interactions; and(f) comparing and analyzing the first and the second analytic values.11. The method of claim 9 or 10, wherein step (c) includes measuring afluorescent signal having a specific wavelength generated by the secondmarkers tagged on second proteins when they are bound to the firstproteins. Here, an optical apparatus generating a near-field is used.12. The method of claim 11, wherein in the step (c), the fluorescentsignal having the specific wavelength is cumulatively measured for apredetermined time period.
 13. The method of claim 11, wherein in thestep (c), the fluorescent signal having the specific wavelength ismeasured in real time under the presence of the cell lysate supplied tothe substrate.
 14. The method of claim 9 or 10, wherein the step (a)includes incubating the cell lysate comprising the first proteins. 15.The method of claim 14, further comprising incubating the substrate witha buffer solution before the step (b).
 16. The method of claim 9,wherein the first marker and the second marker are fluorescent proteinshaving different wavelengths.
 17. An apparatus for analyzing proteininteractions, comprising: a sample loading unit for loading a substratecomprising attached biomolecules which specifically bind to firstmarkers tagged on the first proteins, wherein the first proteins aresupplied to the substrate to induce binding of the biomolecules and thefirst markers, and cell lysate containing the second markers-taggedsecond proteins are supplied to the substrate; an optical excitationunit for generating a near-field having a first wavelength to a surfaceof the substrate loaded on the sample loading unit; and an opticalmeasuring unit for detecting a change from the first wavelength to asecond wavelength on the surface of the substrate according to theinteractions between the first proteins and the second proteins in thecell lysate.
 18. An apparatus of analyzing protein interactions at asingle molecule level, comprising: a sample loading unit for loading asubstrate comprising attached biomolecules which specifically bind tothe first proteins, wherein the first proteins are supplied to thesubstrate and binds to the biomolecules, and cell lysate containing thesecond markers-tagged second proteins are supplied to the substrate; anoptical excitation unit for generating a near-field having a firstwavelength to a surface of the substrate loaded on the sample loadingunit; and an optical measuring unit for detecting a change from thefirst wavelength to a second wavelength on the surface of the substrateaccording to the interactions between the first proteins and the secondproteins in the cell lysate.
 19. The apparatus of claim 17 or 18,wherein the optical measuring unit cumulatively measures a fluorescentsignal having the second wavelength for a predetermined time period. 20.The apparatus of claim 17 or 18, wherein the optical measuring unitmeasures a fluorescent signal having the second wavelength in real timeunder the presence of the cell lysate.
 21. The apparatus of claim 17,wherein the first marker and the second marker are fluorescent proteinshaving different wavelengths.
 22. The apparatus of claim 17 or 18,further comprising a signal analyzing unit for analyzing the fluorescentsignal having the second wavelength measured by the optical measuringunit through image processing.
 23. An apparatus for analyzing proteininteractions, comprising: a sample loading unit for loading a substratecomprising attached biomolecules which specifically bind to firstmarkers tagged on the first proteins, wherein the first proteins aresupplied to the substrate to induce binding of the biomolecules and thefirst markers, and cell lysate containing the second markers-taggedsecond proteins are supplied to the substrate; an optical excitationunit for generating a near-field having a first wavelength to surface ofthe substrate loaded on the sample loading unit; and an opticalmeasuring unit for detecting a change from the first wavelength to asecond wavelength on the surface of the substrate according to theinteractions between the first proteins and the second proteins in thecell lysate; a signal analyzing unit for obtaining the first analyticvalue representing the kinetic picture of the interactions by analyzinga fluorescent signal having the second wavelength measured by theoptical measuring unit through image processing wherein a new substratecomprising attached biomolecules which specifically bind to firstmarkers tagged on the first proteins is loaded on the sample loadingunit, and cell lysate mixture which contains a cell lysate consisting ofthe second markers-tagged second proteins and another cell lysate havingother proteins are supplied to the new substrate, such that the opticalmeasuring unit detects a change from the first wavelength to the secondwavelength on the surface of the substrate according to interactionsbetween the first proteins and the second proteins in the supply of thecell lysate mixture to the new substrate, and the signal analyzing unitobtains a second analytic value by analyzing a fluorescent signal havingthe second wavelength measured by the optical measuring unit throughimage processing.
 24. An apparatus of analyzing interactions betweenfirst proteins and second proteins at a single molecular level,comprising: a sample loading unit for loading a substrate comprisingattached biomolecules which specifically bind to the first proteins,wherein the first proteins are supplied to the substrate and binds tothe biomolecules, and cell lysate containing the second markers-taggedsecond proteins are supplied to the substrate; an optical excitationunit for generating a near-field having a first wavelength to a surfaceof the substrate loaded on the sample loading unit; and an opticalmeasuring unit for detecting a change from the first wavelength to asecond wavelength on the surface of the substrate according to theinteractions between the first proteins and the second proteins in thecell lysate; a signal analyzing unit for obtaining the first analyticvalue representing the kinetic picture of the interactions by analyzinga fluorescent signal having the second wavelength measured by theoptical measuring unit through image processing wherein a new substratecomprising attached biomolecules which specifically bind to firstmarkers tagged on the first proteins is loaded on the sample loadingunit, and cell lysate mixture of a cell lysate consisting of the secondmarkers-tagged second proteins and another cell lysate having otherproteins are supplied to the new substrate, such that the opticalmeasuring unit detects a change from the first wavelength to the secondwavelength on the surface of the substrate according to interactionsbetween the first proteins and the second proteins in the supply of thecell lysate mixture to the new substrate, and the signal analyzing unitobtains a second analytic value by analyzing a fluorescent signal havingthe second wavelength measured by the optical measuring unit throughimage processing.
 25. The apparatus of claim 23 or 24, furthercomprising a signal diagnosing unit for comparing and analyzing thefirst analytic value and the second analytic value.
 26. The apparatus ofclaim 23 or 24, wherein the optical measuring unit cumulatively measuresthe fluorescent signal having the second wavelength for a predeterminedtime period.
 27. The apparatus of claim 23 or 24, wherein the opticalmeasuring unit measures in real time under the presence of the celllysate.
 28. The apparatus of claim 23, wherein the first marker and thesecond marker are fluorescent proteins having different wavelengths.